Para-hydrogen labeled agents and their use in magnetic resonance imaging

ABSTRACT

The invention provides a method of magnetic resonance investigation of a sample, said method comprising: (i) reacting para-hydrogen enriched hydrogen with a hydrogenatable MR imaging agent precursor containing a non-hydrogen non-zero nuclear spin nucleus to produce a hydrogenated MR imaging agent; (ii) administering said hydrogenated MR imaging agent to said sample; (iii) exposing said sample to radiation of a frequency selected to excite nuclear spin transitions of said non-zero nuclear spin nucleus in said hydrogenated MR imaging agent; (v) detecting magnetic resonance signals of said non-zero nuclear spin nucleus from said sample; and (vi) optionally, generating an image or biological functional data or dynamic flow data from said detected signals.

[0001] This invention relates to a method of magnetic resonance imaging(MRI), in particular to non-proton magnetic resonance imaging,especially of nuclei with I (nuclear spin)=½, e.g. ¹³C, ¹⁵N and ²⁹Si.

[0002] Magnetic resonance imaging is a diagnostic technique that hasbecome particularly attractive to physicians as it is non-invasive anddoes not involve exposing the patient under study to potentially harmfulradiation such as X-rays.

[0003] In order to achieve effective contrast between MR images ofdifferent tissue types, it has long been known to administer to thesubject MR contrast agents (e.g. paramagnetic metal species) whichaffect relaxation times in the zones in which they are administered orat which they congregate. By shortening the relaxation times of theimaging nuclei (the nuclei whose MR signal is used to generate theimage) the strength of the MR signal is changed and image contrast isenhanced.

[0004] MR signal strength is also dependent on the population differencebetween the nuclear spin states of the imaging nuclei. This is governedby a Boltzmann distribution and is dependent on temperature and magneticfield strength. However, in MR imaging contrast enhancement has alsobeen achieved by utilising the “Overhauser effect” in which an esrtransition in an administered paramagnetic species is coupled to thenuclear spin system of the imaging nuclei. The Overhauser effect (alsoknown as dynamic nuclear polarisation) can significantly increase thepopulation difference between excited and ground nuclear spin states ofthe imaging nuclei and thereby amplify the MR signal intensity. Most ofthe Overhauser contrast agents disclosed to date are radicals which areused to effect polarisation of imaging nuclei in vivo. There is verylittle reported work on techniques which involve ex vivo polarisation ofimaging nuclei prior to administration and MR signal measurement.

[0005] U.S. Pat. No. 5,617,859 (Souza) discloses a magnetic resonanceimaging system employing a small, high-field polarizing magnet (e.g. a15T magnet) to polarize a frozen material which is then warmed up andadministered to a subject placed within the imaging apparatus. Thematerial used may be water, saline, a fluorocarbon or a noble gas suchas He or Xe. Since the magnetic field in the polarizing magnet isgreater than that inside the imaging apparatus and since polarization iseffected at low temperature, an increased population difference betweenthe nuclear spin states (i.e. polarization) should result in a strongerMR signal from the polarized material.

[0006] In U.S. Pat. No. 5,611,340 (Souza), a somewhat similar MR imagingsystem is disclosed. Here however liquid hydrogen is polarized by thepolarizing magnet and thereafter it is heated up and reacted with oxygento produce polarized water which is administered to the subject. Theresulting enhanced MR signal will be an enhanced ¹H MR signal.

[0007] U.S. Pat. No. 5,545,396 (Albert) discloses an in vivo MR imagingmethod in which a noble gas (e.g. ¹²⁹Xe or ³He) having a hyperpolarisednuclear spin is inhaled into the lungs and a representation of itsspatial distribution therein is generated. MR imaging of the human oralcavity using hyperpolarised ¹²⁹Xe was also reported by Albert in J. Mag.Res., 1996: B111, 204-207.

[0008] The use of hyperpolarised MR contrast agents in MR investigationssuch as MR imaging has the advantage over conventional MR techniques inthat the nuclear polarisation to which the MR signal strength isproportional is essentially independent of the magnetic field strengthin the MR apparatus. Currently the highest obtainable field strengths inMR imaging apparatus are about 8T, while clinical MR imaging apparatusare available with field strengths of about 0.2 to 1.5T. Sincesuperconducting magnets and complex magnet construction are required forlarge cavity high field strength magnets, these are expensive. Using ahyperpolarised contrast agent, since the field strength is less criticalit is possible to make images at all field strengths from earth field(40-50 μT) up to the highest achievable fields. However there are noparticular advantages to using the very high field strengths where noisefrom the patient begins to dominate over electronic noise (generally atfield strengths where the resonance frequency of the imaging nucleus is1 to 20 MHz) and accordingly the use of hyperpolarised contrast agentsopens the possibility of high performance imaging using low cost, lowfield strength magnets.

[0009] The present invention is based on a method of MRI of a samplewhich relies on ex vivo nuclear polarisation of selected non-hydrogen,I≠0 imaging nuclei (e.g. ¹³C, ¹⁵N and ²⁹Si nuclei) of an MR imagingagent by reaction of a precursor to said agent with para-hydrogenenriched hydrogen gas.

[0010] Thus viewed from one aspect the present invention provides amethod of magnetic resonance investigation of a sample, preferably ahuman or non-human animal body (e.g. a mammalian, reptilian or avianbody), said method comprising:

[0011] (i) reacting para-hydrogen enriched hydrogen with ahydrogenatable MR imaging agent precursor containing a non-zero nuclearspin nucleus other than ¹H to produce a hydrogenated MR imaging agent;

[0012] (ii) administering said hydrogenated MR imaging agent to saidsample;

[0013] (iii) exposing said sample to radiation of a frequency selectedto excite nuclear spin transitions of said non-zero nuclear spin nucleusin said hydrogenated MR imaging agent;

[0014] (iv) detecting magnetic resonance signals of said non-zeronuclear spin nucleus from said sample; and

[0015] (vi) optionally, generating an image or biological functionaldata or dynamic flow data from said detected signals.

[0016] The MR signals obtained in the method of the invention may beconveniently converted into 2- or 3-dimensional image data or intofunctional, flow or perfusion data by conventional manipulations.

[0017] Hydrogen molecules exist in two different forms, namelypara-hydrogen (p-H₂) where the nuclear spins are antiparallel and out ofphase (the singlet state) and ortho hydrogen (o-H₂) where they areparallel or antiparallel and in phase (the triplet state) At roomtemperature, the two forms exist in equilibrium with a 1:3 ratio ofpara:ortho hydrogen. At 80K the ratio is 48:52 and at 20K it approaches100:0, i.e. 99.8:0.2. Reducing the temperature still further is notbeneficial since hydrogen freezes at about 17K. The rate ofequilibration is very low in pure hydrogen but in the presence of any ofseveral known catalysts (such as Fe₃O₄, Fe₂O₃, or activated charcoal) anequilibrium mixture is rapidly obtained and remains stable at roomtemperature for several hours after separation from the catalyst. Thusby “enriched hydrogen” above is meant hydrogen in which there is ahigher than equilibrium proportion of para-hydrogen, for example wherethe proportion of para-hydrogen is more than 25%, preferably more than30%, preferably 45% or more, more preferably 60% or more, particularlypreferably 90% or more, especially preferably 99% or more. Typically thepreparation of enriched hydrogen, an optional initial step in the methodaccording to the invention, will be carried out catalytically at lowtemperatures e.g. at 160K or less, preferably at 80K or less or morepreferably at about 20K.

[0018] The parahydrogen thus formed may be stored for long periods,preferably at low temperature, e.g. 18-20° K. Alternatively it may bestored in pressurized gas form in containers with non-magnetic andnon-paramagnetic inner surfaces, e.g. a gold or deuterated polymercoated container.

[0019] Generally speaking, if a p-H₂ molecule is transferred to a hostmolecule by means of catalytic hydrogenation (optionally at elevatedpressure (e.g. 50 to 100 bar)), the proton spins remain antiparallel andbegin to relax to thermal equilibrium with the normal time constant T₁of the hydrogen in the molecule (typically about one second). Howeverduring relaxation some of the polarisation may be transferred toneighbouring nuclei by cross-relaxation or other types of coupling. Thepresence of, for example, a ¹³C nucleus with a suitable substitutionpattern close to the relaxing hydrogen may lead to the polarisationbeing conveniently trapped in the slowly relaxing ¹³C nucleus. Anenhancement factor of 2580 has been reported in the literature(Barkemeyer et al, 1995, J Am Chem Soc 117, 2927-2928). A ¹³C nucleus ina carbonyl group or in certain quaternary carbons may have a T1relaxation time typically of more than a minute.

[0020] The hydrogenation step should preferably be performed in theliquid or gaseous phase, preferably in the absence of materials whichwould promote relaxation. If in the liquid phase, then the catalyst canbe removed by filtration through, for example, an ion-exchange resin. Ifin the gas phase, then separation of a solid catalyst is trivial and theMR imaging agent formed can simply be passed into a suitable solvent,preferably a physiologically tolerable solvent, most preferably water,and used according to the invention.

[0021] Thus the present invention is based on the recognition thatpolarisation of certain nuclei (e.g. ¹³C nuclei) in a host moleculeusing enriched hydrogen represents a means for performing ex vivopolarisation of an MR imaging agent prior to its administration into asubject and conventional MR imaging. The term “MR imaging agent” usedherein refers to an agent containing nuclei (MR imaging nuclei) capableof emitting magnetic resonance signals. Such MR imaging nuclei arenon-zero nuclear spin nuclei capable of emitting magnetic resonancesignals, preferably I=½ nuclei (other than hydrogen itself), such ase.g. ¹⁹F, ³Li, ¹H, ¹³C, ¹⁵N, ²⁹Si or ³¹P nuclei, but preferably are ¹³Cor ¹⁵N nuclei, most preferably ¹³C nuclei. In other words, the MRimaging agent precursor should preferably contain a non-hydrogen I=½nucleus.

[0022] The non hydrogen non zero nuclear spin nucleus in the MR imagingagent may be present in its naturally occurring isotopic abundance.However where the nucleus is a non-preponderant isotope (e.g. ¹³C where¹²C is the preponderant isotope) it will generally be preferred that thecontent of the nucleus be enriched, ie. that it is present at a higherthan normal level.

[0023] Thus viewed from a further aspect the present invention providesthe use of hydrogen (e.g. para-hydrogen enriched hydrogen) in MR imagingof a sample (e.g. a human body), preferably ¹³C, or ¹⁵N MR imaging of asample.

[0024] Viewed from an alternative aspect, the invention provides the useof para-hydrogen enriched hydrogen for the manufacture of an MR imagingagent for use in a method of diagnosis involving generation of an MRimage by non ¹H MR imaging of a human or non-human animal body.

[0025] Viewed from a still further aspect the invention provides use ofa hydrogenatable compound containing a non hydrogen non-zero nuclearspin nucleus in the manufacture of an MR imaging agent for use in amethod of diagnosis involving generation of an MR image by non-proton MRimaging, said manufacture involving hydrogenation of said compound withpara-hydrogen enriched hydrogen.

[0026] By imaging, it will be appreciated that not just production oftwo or three dimensional morphological images is covered: the imagesproduced may be representations of the value or temporal change in valueof a physiological parameter such as temperature, pH, oxygen tension,etc. Morphological images however will generally be produced.

[0027] MR imaging agent precursors suitable for use in the presentinvention are hydrogenatable and will typically possess one or moreunsaturated bonds, e.g. double or triple carbon-carbon bonds. For invivo imaging, the hydrogenated MR imaging agent should of course bephysiologically tolerable or be capable of being presented in aphysiologically tolerable form.

[0028] The MR imaging agent should preferably be strongly polarisable(for example, to a level of greater than 5%, preferably greater than10%, more preferably greater than 25%) and have a non-hydrogen MRimaging nucleus with a long T₁ relaxation time under physiologicalconditions, e.g. ¹³C, ¹⁵N or ²⁹Si. By a long T₁ relaxation time is meantthat T₁ is such that once polarised, the MR imaging agent will remain sofor a period sufficiently long to allow the imaging procedure to becarried out in a comfortable time span. Significant polarisation shouldtherefore be retained for at least is, preferably for at least 60 s,more preferably for at least 100 s and especially preferably 1000 s orlonger.

[0029] There will preferably be nuclear spin:spin coupling in theimaging agent between the MR imaging nucleus and at least one of thehydrogens introduced as a result of hydrogenation with para-hydrogen.The coupling constant is preferably between 1 and 300 Hz, morepreferably between 10 and 100 Hz. This is preferably achieved by placingthe MR imaging nucleus no more than 3, more preferably no more than 2bonds away from the para-hydrogen derived hydrogen. Desirably the nmrsignal from the MR imaging nucleus (hereinafter occasionally referred toas the reporter nucleus), is sharp, preferably with a linewidth (at 37°C. in blood or tissue) of less than 100 Hz, more preferably less than 10Hz, even more preferably less than 1 Hz. Accordingly, the MR imagingagent will preferably contain as few non-zero nuclear spin atoms(besides the reporter nucleus and the two protons from the p.H₂) aspossible which can couple with the reporter nucleus. Desirably thereforethe MR imaging agent contains no more than 10, more preferably no morethan 5, still more preferably no more than 2, even more preferably nomore than 1, and especially preferably no non-zero nuclear spin nucleiwithin 3 bonds of the reporter nucleus, and still more preferably within4 bonds. Most preferably the only non-zero nuclear spin nuclei in the MRimaging agent are the reporter nucleus and the protons from the p.H₂.Quadrupolar nuclei (e.g. ¹⁴N, ³⁵Cl and ⁷⁹Br) should preferably not beincluded in the MR imaging agent although they may be present incounterions or other dissolved components of a contrast mediumcontaining the MR imaging agent. Avoidance of undesired nuclei mayinvolve use of deuterium in place of protons in the MR imaging agent.Thus where the unsaturated bond to be hydrogenated is a C═C bond, thismay desirably be in a —CD=CD- structure. In this way the polarizationtransfer to the reporter nucleus, e.g. ¹³C in a —¹³C—C═C— structure maybe increased. The MR imaging agent should preferably be relatively small(e.g. molecular weight less than 500D, more preferably less than 300D(e.g. 50-300D) and more preferably 100 to 200D) and also preferablyshould be soluble in a liquid solvent or solvent mixture, mostpreferably in water or another physiologically tolerable solvent orsolvent mixture. The MR imaging agent precursor likewise is preferablysoluble in such solvents or solvent mixtures and desirably is capable ofundergoing rapid catalysed hydrogenation, e.g. at a conversion rate ofat least 1 g precursor/min using 2 mole % or less of catalyst.Furthermore, the chemical shift, or even better the coupling constant ofthe nmr signal from the imaging nucleus in the MR imaging agent shouldpreferably be influenced by physiological parameters (e.g. morphology,pH, metabolism, temperature, oxygen tension, calcium concentration,etc). For example, influence by pH can be used as a general diseasemarker, whilst influence by metabolism may be a cancer marker.Alternatively, the MR imaging agent may conveniently be a material whichis transformed (e.g. at a rate such that its half life is no more than10×T₁ of the reporter nucleus, preferably no more than 1×T₁) in thesubject under study to a material in which the reporter nucleus has adifferent coupling constant or chemical shift. In this case the subjectmay be inanimate or animate, e.g. a human or animal, a cell culture, amembrane-free culture, a chemical reaction medium, etc. Thus for examplethe reporter nucleus may provide information on the operation of thebiochemical machinery of an organism where that machinery transforms theMR imaging agent and in so doing changes the chemical shift or couplingconstant of the reporter nucleus. It will be appreciated that theimaging process used in this case may be an nmr spectroscopic procedurerather than (or in addition to) an imaging procedure which generates amorphological image.

[0030] The MR imaging agent should preferably be ¹³C or ¹⁵N enriched,particularly preferably ¹³C enriched, in positions close to thehydrogenation site, e.g. a double or triple bond, and where relaxationis slow. Preferred MR imaging agents according to the invention alsoexhibit the property of low toxicity.

[0031] Generally speaking, to increase the MR signal from thehydrogenated MR imaging agent, it may be desirable to incorporate morethan one unsaturated bond in each molecule of the precursor, e.g. in aconjugated unsaturated system. However due consideration must be givento the need to keep molecular weight relatively low to preventdifficulties in administration of the agent. The presence of one or moreC≡C bonds in the hydrogenatable MR imaging agent precursor increases thereaction rate and may therefore be preferred. Also preferred arecompounds with an unsaturated carbon-carbon bond with one or morecarbonyl substituents, e.g. an αβ unsaturated carbonyl compound.Particularly preferred are compounds comprising disubstitutedunsymmetric alkylene or acetylene groups with acarbonyl-unsaturation-carbonyl moiety. Such compounds are of highreactivity and may allow two or more ¹³C atoms to be incorporated toutilize the polarisation more efficiently.

[0032] Precursors that match as many of the above design parameters aspossible have been found to form excellent MR imaging agents oncereacted with parahydrogen. Such agents have both in vitro and in vivousage.

[0033] Such MR imaging agents and their precursors which are reporternucleus enriched, ie. have greater than natural isotopic abundance ofthe reporter nucleus, are novel and form further aspects of theinvention. Viewed from a first of these aspects the invention provides aprecursor compound:

[0034] (i) containing a hydrogenatable unsaturated bond;

[0035] (ii) containing a non-hydrogen non zero nuclear spin nucleus atgreater than natural isotopic abundance;

[0036] (iii) having a molecular weight preferably below 1000D, morepreferably below 500D; and

[0037] (iv) which following hydrogenation has an nmr spectrum for saidnon-hydrogen non zero nuclear spin nucleus which is a multiplet having acoupling constant relative to one of the hydrogens introduced byhydrogenation of 1 to 300 Hz and having a linewidth of less than 100 Hz,preferably below 10 Hz, more preferably below 1 Hz.

[0038] The hydrogenatable precursor compound of the invention preferablycontains as said non-hydrogen non zero nuclear spin nucleus a I=½nucleus such as ¹³C, ¹⁵N or ²⁹Si, especially ¹³C. Preferably it also hassome or all of the desired properties discussed earlier, e.g.solubility, pavcity of other I-0 nuclei (although these may be presentin a counterion component of the compound if it is ionic), reactivity tohydrogenation, etc.

[0039] Viewed from a further aspect the invention also provides areporter compound:

[0040] (i) containing at least two protons;

[0041] (ii) containing a non-hydrogen non zero nuclear spin nucleus atgreater than natural isotopic abundance;

[0042] (iii) having a molecular weight preferably below 1000D, morepreferably below 500D; and

[0043] (iv) having an nmr spectrum for said non-hydrogen non zeronuclear spin nucleus which is a multiplet having a coupling constantrelative to one of said at least two protons 1 to 300 Hz and having alinewidth of less than 100 Hz, preferably below 10 Hz, more preferablybelow 1 Hz.

[0044] Once again, the reporter compounds of the invention, which areobtainable by hydrogenation of the precursor compounds of the inventionwill desirably possess some or all of the desired properties referred toearlier, e.g. solubility, narrow linewidths, coupling constants in the10 to 100 Hz range, coupling constant sensitivity, chemical shiftsensitivity, isotopic make up, etc.

[0045] Preferred precursor compounds for MR imaging agents for useaccording to the invention desirably contain the following molecularsub-units:

[0046] (i) at least one C═C or C≡C bonds;

[0047] (ii) a C, N or Si atom separated by one or two bonds from a C═Cor C≡C bond, bound only to atoms the naturally most abundant isotopeform of which has a nuclear spin I=0, and not coupled by a series ofcovalent bonds to any atoms the naturally most abundant isotopic form ofwhich has I>½; and

[0048] (iii) at least one water-solubilizing moiety, ie. a functionalgroup which imparts water solubility to the molecule, e.g. hydroxyl,amine or oxyacid (e.g. carboxyl) groups.

[0049] Correspondingly, preferred MR imaging agents for use according tothe invention desirably contain the following molecular sub-units:

[0050] (i) at least one CH—CH or CH═CH moiety;

[0051] (ii) a C, N or Si atom separated by one or two bonds from a CH—CHor CH═CH moiety, bound only to atoms the naturally most abundantisotopic form of which has I=0, and not coupled by a series of covalentbonds to any atoms the naturally most abundant isotopic form of whichhas I>½; and

[0052] (iii) at least one water-solubilizing moiety, ie. a functionalgroup which imparts water solubility to the molecule, e.g. hydroxyl,amine or oxyacid (e.g. carboxyl) groups.

[0053] While compounds meeting these criteria can be used according tothe invention without enrichment in ¹³C, ¹⁵N or ²⁹Si, it is preferredthat they be enriched and in particular that there be such isotopicenrichment of the atoms defined by criterion (ii).

[0054] Specifically preferred hydrogenatable MR imaging agent precursorsfor use in the method of the invention include simple unsaturated acids(e.g. acrylic acid, crotonic acid, propionic acid, fumaric acid, maleicacid and HOOC.C≡C.COOH), especially where a carboxyl carbon separated bytwo or more favourably one bond from the unsaturated bond is ¹³C or ¹³Cenriched,

[0055] unsaturated quaternary carbon compounds where the quaternarycarbon is separated by two or more preferably one bond from theunsaturated bond and preferably where the quaternary carbon is ¹³C or¹³C enriched, e.g.

[0056] compounds with more than one hydrogenation site such as

[0057] especially where a carbon separated by two or more preferably onebond from an unsaturated bond is ¹³C or ¹³C enriched and other compoundssuch as:

[0058] (where R₁ is

[0059] R₃ is alkyl, hydroxyalkyl, amino, hydroxyl etc, R is CONHR₂ andR₂ is a conventional hydrophilic group known to be useful in X-raycontrast media such as a straight chain or branched C₁₋₁₀-alkyl group,preferably a C₁₋₅ group, optionally with one or more CH₂ or CH moietiesreplaced by oxygen or nitrogen atoms and optionally substituted by oneor more groups selected from oxo, hydroxy, amino, carboxyl derivative,and oxo substituted sulphur and phosphorus atoms).

[0060]¹³C enriched MR imaging agents have ¹³ C at one particularposition (or more than one particular position) in an amount in excessof the natural abundance, i.e above about 1%. Preferably such a singlecarbon position will have 5% or more ¹³C, particularly preferably 10% ormore, especially preferably 25% or more, more especially preferably 50%or more, even more preferably in excess of 99% (eg 99.9%).

[0061] In all these hydrogenatable compounds represented by formulaeherein, protons (H) are preferably replaced by deuterons, except perhapsprotons which are labile on dissolution (e.g. carboxyl protons).

[0062] In addition, compounds which on hydrogenation yield compoundswhich are or are analogous to naturally occurring biomolecules (e.g.amino acids, metabolites, neurotransmitters, etc) are possible MRimaging agent precursors for use in the method of the invention.

[0063] For studies of biochemical reactions, it may also be interestingto use succinic acid (which occurs in the citric acid cycle), especially¹³C enriched succinic acid:

[0064] For studies of peptide/protein synthesis, whether natural orartificial, it may likewise be interesting to use amino acids, producedby pH_(z) hydrogenation of a β carbon-γ carbon unsaturated bond,especially where the carboxyl carbon is ¹³C enriched.

[0065] Amides, amines, cyanides and nitroxides or other nitrogencontaining MR imaging agents are particularly suitable for ¹⁵N reporternuclei as are compounds which comprise a ring nitrogen containingheterocycle. One example of a ¹⁵N reporter nucleus imaging agent isacetyl choline, which is biologically modified and so may be used tostudy metabolic processes. This imaging agent may be produced by pH₂hydrogenation of corresponding ethylenically or acetylenicallyunsaturated precursors, preferably ones enriched in ¹⁵N:

[0066] Likewise amino acids, especially deuterated versions thereof maybe used as vehicles for ¹⁵N. Silane and silicone compounds may similarlybe used as vehicles for ²⁹Si.

[0067] Due to their biotolerability, compounds with quaternary carbonsmay be preferred. Cationic compounds may also be used, e.g. quaternaryammonium salts.

[0068] One especially preferred hydrogenatable or hydrogenated MRimaging agent is maleic acid dimethyl ester which is the hydrogenationproduct of acetylene dicarboxylic acid dimethyl ester.

[0069] Another useful MR imaging agent would be methionine, and thus anunsaturated methionine precursor may advantageously be used as theprecursor compound.

[0070] Other interesting precursors include acetylenic compounds such asthe following

[0071] where R is H or C₁₋₆ alkyl and R^(i) is hydroxylalkyl, or asulphone or sulphoxide.

[0072] Typically the hydrogenatable MR imaging agent precursor willundergo hydrogenation in the presence of a suitable catalyst, optionallyat elevated temperature or pressure. The hydrogenation catalyst used inthe method of the invention need not be a homogeneous catalyst butduring hydrogenation the entire hydrogen molecule should be transferredto the host molecule. Some examples of catalysts that are able to fulfilthis criterion are shown in Table 1. TABLE 1 Hydrogenation catalyststhat transfer dihydrogen to a double or triple bond Water Solu- Com-Catalyst Synonym bility ment (PPh₃)RhCl Wilkinson's − Active whencatalyst bound to zeo- lite (12Å) [(NBD)Rh + Cationic (Amphos)₂]³⁺(TPPMS)₃RhCl + Anionic (HEXNa)₂RhCl + Anionic (OCTNa)₂RhCl + AnionicIrCl(CO)(PPh₃)₂ Vasca's complex − (bicyclo[2.2.1]hep ta-2,5-diene)[1,4-bis(diphenylphosphino)butane] rhodium(I) tetrafluoroborate

[0073] It has been found that rhodium catalysts are particularly usefulin the hydrogenation step, most particularly those rhodium catalystscomprising phosphine groups.

[0074] The reaction mechanism of hydrogenation of ethylene withWilkinson's catalyst is shown by way of example in FIG. 2. Reversibilityof reaction is found to be low with such catalysts containing cyclicphosphines.

[0075] A further catalytic cycle is shown by way of example in FIG. 3.The oxidative addition of enriched hydrogen to the catalyst is generallyan equilibrium step which means that certain catalysts will alsointerconvert p-H₂ and o-H₂. It is therefore desirable that the chosenhydrogenatable MR imaging agent precursor is highly reactive.

[0076] It is highly desirable to carry out the hydrogenation step in avery low magnetic field. Preferably this very low magnetic field islower than the magnetic field of the earth itself, that is lower than 50μT, more preferably less than 10 μT, even more preferably less than 2μT, e.g. 0 to 1 μT, especially 0.3 to 1 μT. It is possible to createsuch low magnetic fields using, for example, commercially availablemagnetic shielding, for example μ-metal. The effect of the magneticfield on the degree of polarization of a reporter nucleus (in this casea ¹³C nucleus) is shown in FIG. 1.

[0077] It will be apparent that the degree of solubility of thehydrogenated MR imaging agent will determine how rapidly it can bedissolved in administrable media and subsequently administered and,given the finite lifetime of the polarisation, the importance of thesefactors will be clear. Thus hydrogenation is conveniently performed inaqueous media and preferred catalysts for use in the invention shouldoperate efficiently in water and conveniently not facilitate theexchange of hydrogen atoms between water and the enriched hydrogen,otherwise the polarisation is quickly lost. A water soluble rhodiumcatalyst is one preferred example.

[0078] In order to facilitate rapid separation of catalyst andhydrogenated MR imaging agent after hydrogenation, the catalyst maypreferably be one which is immobilized on a solid material e.g. apolymeric material which allows the catalyst-bound solid material to berapidly filtered off after reaction. Known examples useful for thepresent method include catalysts covalently linked to a support oradsorbed on silica.

[0079] An alternative way to remove catalyst from an aqueous solution isto run the reaction in the presence of a water-soluble catalyst (e.g. arhodium catalyst) which may then be removed by filtration through anion-exchange resin or any other sort of filter that can retain thecatalyst and allow the product to pass. In the preferred case of acationic catalyst, filtration may be carried out through a cationexchanger. Particularly preferred catalysts are cationic rhodiumcatalysts Rhodium catalysts transfer hydrogen as a unit to one substratemolecule and therefore avoid problems of H₂/D₂ scrambling. One suchembodiment makes use of an ion-exchange resin bound cationic complexsuch as [(NBD)Rh(Amphos)₂]³⁺. The aqueous solution of an anionic orneutral product is obtained-in the filtrate. The opposite procedure mayof course be used for anionic catalysts but these are generally lesspreferred. A neutral catalyst may be separated from the MR imaging agentby making use of physical characteristics such as lipophilicity. Forexample, a lipophilic catalyst (e.g. Wilkinson's catalyst) may be usedin a biphasic system such as water/C18-derivatised silica or even twoimmiscible liquids such as water/heptane.

[0080] Hydrogenation may take place advantageously in a non-aqueousmedia in which the hydrogenation product is insoluble (i.e. from whichit precipitates). The increased T₁ of the solid MR imaging agent allowsmore time for isolation and subsequent dissolution in an administrablemedium. Hydrogenation may also take place with the MR imaging agentprecursor being insoluble in non-aqueous media but with a particle sizeas small as possible to increase relative surface area. The use ofnon-aqueous media, preferably media with non-magnetically active nuclei(e.g. CS₂ or CO₂ under supercritical conditions) advantageously reducespolarisation loss from the polarised MR imaging agent and allows the useof an extended range of catalysts.

[0081] Viewed from another aspect the invention provides apparatus forhydrogenation comprising:

[0082] a reaction chamber having therein a reaction zone, said reactionchamber having a gas inlet and a gas outlet;

[0083] a temperature controller arranged to control the temperature insaid reaction zone; and

[0084] magnetic shielding arranged about said reaction zone andsufficient to cause the magnetic field in said reaction zone to be lessthan 10 μT, preferably less than 1 μT.

[0085] The reaction chamber will conveniently be disposed within agenerally cylindrical μ-metal shield. This shield preferably has severalconcentric layers, e.g. a μ-metal layer of relatively high permittivitysurrounded by a demagnetizing layer, e.g. of copper foil, and in turnsurrounded by one or more layers of μ-metal of lower permittivity thanthe inner layer. The inner μ-metal layer is preferably of μ-metal of thehighest available permittivity.

[0086] At each axial end, the cylindrical magnetic shield preferablyextends in its axial direction beyond the reaction zone by at least theinternal diameter of the shield. Although a circular cross-section ispreferred, the cylindrical shield may be non-circular in cross-section,e.g. elliptical or polygonal, for example hexagonal. Where thecross-section is non-circular, the axial extension beyond the reactionzone is preferably by at least the minimum internal “diameter” (e.g.face to face spacing for a hexagonal cross section) but more preferablyby at least the maximum internal diameter (e.g. corner to corner spacingfor a hexagonal cross section).

[0087] The reaction zone may be for example comprise a bed of beadsthrough which hydrogen may flow upwards from a lower gas inlet andthrough which a solution containing hydrogenatable precursor andhydrogenation catalyst may pass down to be removed from the reactionchamber through a lower product outlet. Alternatively, the beads mayhave the catalyst immobilized thereon so that the product solution iscatalyst free and may be in a form ready to use. The beads arepreferably formed from paramagentic material free polymer, glass orsilica or are of a non-magnetic metal. Selection of bead size (e.g. 0.5to 5 mm diameter, preferably 2 mm), bed depth and choice of direction ofhydrogen flow will determine the duration of the reaction (generally 10to 60 sec.). The preferred duration and bed depth can be determined byroutine experimentation for the selected precursor/catalyst combination.

[0088] The temperature controller will conveniently be a heating/coolingjacket disposed about the reaction zone portion of the reaction chamberand within the shield. Preferably the materials used are non magnetic. Awater- or gas-jacket is generally appropriate. A temperature sensor maybe disposed in or adjacent the reaction zone if desired.

[0089] Conveniently, the reaction chamber has a precursor solution inletabove the reaction zone and an MR imaging agent solution outlet belowthe reaction zone. Thus in operation using this embodiment the followingactions are performed:

[0090] a source of pH₂ enriched hydrogen is attached to the gas inlet;

[0091] the reaction chamber is flushed with the enriched hydrogen;

[0092] water of the desired temperature is flowed through thewater-jacket;

[0093] a quantity of a solution, preferably a sterile aqueous solution,of the precursor compound is introduced into the reaction chamber andinto a particulate bed through which the enriched hydrogen is flowingupwardly; and

[0094] the solution passing out of the bed is withdrawn from thereaction chamber, optionally after reversal of hydrogen flow directionto drive the solution out of the bed.

[0095] Where the catalyst is not immobilized on the particles of thebed, it will generally be included in the precursor solution, either indissolved or particulate or supported form. If desired the catalyst maybe removed from the product solution, e.g. by precipitation and/orfiltration or by passage over a material (e.g. an ion exchange column orlipophilic surface) which has affinity for the catalyst.

[0096] Catalyst removal clearly depends on the nature of the catalyst,the precursor, the MR imaging agent and whether the subject to be imagedis a living human or animal or not. Thus for inanimate subjects,catalyst removal may be unnecessary. In one embodiment a hydrogenationcatalyst soluble in a solvent that is imiscible with water is used andthe hydrogenation reaction is carried out in water with a substrate thatis soluble in organic solvents but has a distribution constant thatfavours water. The substrate is extracted into water that is injectedi.v. In another embodiment a water-soluble polymer bound hydrogenationcatalyst is used and the hydrogenation reaction is performed in waterwith a water-soluble substrate. The catalyst is removed by filtrationprior to i.v. injection. In a third embodiment a solid polymer-boundhydrogenation catalyst is used and the hydrogenation reaction isperformed in water with a water-soluble substrate. The catalyst isremoved by filtration prior to i.v. injection. In a fourth embodiment asolid polymer-bound hydrogenation catalyst is used and the hydrogenationreaction is performed in water with a water-soluble substrate. Thecatalyst is removed by filtration prior to i.v. injection.

[0097] The withdrawal of the product solution is preferably by passagethrough a valve into the barrel of a syringe. The syringe may then beused to administer the MR imaging agent, e.g. by injection into a humanor animal subject. The inner walls of the syringe and indeed of anyapparatus components contacted by the hydrogenated MR imaging agent arepreferably substantially free of paramagnetic (and ferro andferrimagnetic) materials. Likewise the period of contact of the MRimaging agent with any surfaces between hydrogenation and administrationshould preferably be kept to a minimum.

[0098] In a preferred embodiment, the apparatus of the inventioncomprises:

[0099] (i) a reservoir of enriched hydrogen, preferably cooled, e.g. toliquid form;

[0100] (ii) a reaction chamber having a reaction zone containing aparticulate bed and having a first gas inlet below said bed, a first gasoutlet above said bed, a solution inlet above said bed and a solutionoutlet below said bed, and preferably a second gas inlet above said bedand optionally a second gas outlet below said bed (optionally since thesolution outlet may function as a gas outlet);

[0101] (iii) a gas conduit from said reservoir to said first gas inletin the reaction chamber, optionally provided with a heater to raise thetemperature of gas flowing therethrough, and optionally provided with avalve to direct gas flow to said second gas inlet rather than to saidfirst gas inlet;

[0102] (iv) a temperature controller, e.g. a water or gas jacket,disposed around said reaction chamber at at least said reaction zone;and

[0103] (v) a magnetic shield disposed around said reaction chamber at atleast said reaction zone.

[0104] The inlets and outlets to the reaction chamber are eachpreferably provided with a valve or if appropriate a septum and meansfor attaching vessels, e.g. the hydrogen reservoir, a syringe forreceiving the MR imaging agent, a syringe containing the precursorsolution, and reservoirs for receiving exhaust hydrogen (for recycling).

[0105] Such an apparatus may be set up near the MR imaging apparatus,e.g. so that the imaging agent may be manufactured “on-site” usingreservoirs of pH₂ enriched hydrogen supplied from the, normally distant,location where the enriched hydrogen was prepared.

[0106] Alternatively, the apparatus may be arranged for a gas phasereaction with precursor and hydrogen being introduced into the reactionzone in gas form and with the exhaust gas being cooled to separatehydrogen (which will remain gaseous), precursor, MR imaging agent, and,the hydrogenation catalyst. With different boiling points, the imagingagent, precursor and if appropriate, the catalyst may be collectedseparately and removed for optional formulation (e.g. dissolution in anappropriate liquid medium) and administration in the case of the MRimaging agent and for recycling or subsequent reuse in the case of othercomponents. The catalyst could be immobilized on a surface (e.g. thesurface of beads in a bed or of capillaries in a bundle of parallelcapillaries) or could be included in the gas flow as a gas or asentrained droplets or particles. To ensure adequate progression of thereaction, the reaction zone could be arranged in a spiral or the likewithin the magnetic shield and the reaction can be performed at elevatedtemperature and pressure. Apparatus comprising shielding, reactionchamber, temperature controller, gas inlets, MR imaging agent separator(e.g. a condenser) and gas outlet arranged for performing thehydrogenation in the gas phase forms a further aspect of the invention.

[0107] In one embodiment of the method of the invention, the polarised(hydrogenated) MR imaging agent may be stored at low temperature e.g. infrozen form. Generally speaking, at low temperatures the polarisation isretained longer and thus polarised MR imaging agents may conveniently bestored e.g. in liquid nitrogen. Prior to administration, the MR imagingagent may be rapidly warmed to physiological temperatures usingconventional techniques such as infrared or microwave radiation.

[0108] Viewed from a further aspect the invention provides aphysiologically tolerable MR imaging agent composition comprising an MRimaging agent together with one or more physiologically tolerablecarriers or excipients, said imaging agent containing nuclei of anon-hydrogen I=½ isotope (e.g. ¹³C, ¹⁵N or ²⁹Si), preferably at a higherthan natural abundance, characterised in that said nuclei are polarizedsuch that their nmr signal intensity is equivalent to a signal intensityachievable in a magnetic field of at least 0.1T, more preferably atleast 25T, particularly preferably at least 100T, especially preferablyat least 450T, e.g. at 21° C. in the same composition. Preferably thecomposition is sterile and is stable at a physiologically tolerabletemperature (e.g. at 10-40° C.).

[0109] Polarization is given by the equation$P = {\frac{{N\quad \alpha} - {N\quad \beta}}{{N\quad \alpha} + {N\quad \beta}}}$

[0110] which at equilibrium is equal to$\frac{1 - {\exp \left( {{- {\gamma\hslash}}\quad {B_{o}/{kT}}} \right)}}{1 + {\exp \left( {{- {\gamma\hslash}}\quad {B_{o}/{kT}}} \right)}}$

[0111] where Nα is the number of spins in nuclear spin state α (e.g.+½);

[0112] N_(β) is the number of spins in nuclear spin state β (e.g. −½);

[0113] γ is the magnetogyric ratio for the isotopic nucleus in question,e.g. ¹³C);

[0114]

is Planck's constant divided by 2n;

[0115] B_(o) is the magnetic field;

[0116] k is Boltzmann's constant; and

[0117] T is temperature in kelvin.

[0118] Thus P has a maximum value of 1 (100% polarization) and a minimumvalue of 0 (0% polarization). For ¹³C the maximum polarizationobtainable by the low-field para-hydrogen hydrogenation methodcorresponds to about 0.5 MT.

[0119] Given that the method of the invention should be carried outwithin the time that the MR imaging agent remains significantlypolarised, once hydrogenation has occurred and if desired or necessarythe catalyst has been removed, it is desirable for administration of theMR imaging agent to be effected rapidly and for the MR measurement tofollow shortly thereafter. This means that the sample (e.g. body ororgan) should be available close to the area in which the polarisationhas been carried out. If this is not possible, the material should betransported to the relevant area at low temperature.

[0120] The preferred administration route for the MR imaging agent isparenteral, e.g. by bolus injection, by intravenous or intra-arterialinjection or, where the lungs are to be imaged, by spray, e.g. byaerosol spray. Oral and rectal administration may also be used.

[0121] Where the MR imaging nucleus is other than a proton (e.g. ¹³C),there will be essentially no interference from background signals (thenatural abundance of ¹³C, ¹⁵N, ²⁹Si etc. being negligible) and imagecontrast will be advantageously high. Thus the method according to theinvention has the benefit of being able to provide significant spatialweighting to a generated image. In effect, the administration of apolarised MR imaging agent to a selected region of a sample (e.g. byinjection) means that the contrast effect is, in general, localised tothat region. The precise effect of course depends on the extent ofbiodistribution over the period in which the MR imaging agent remainssignificantly polarised. In general, specific body volumes (i.e. regionsof interest such as the vascular system) into which the MR imaging agentis administered may be defined with improved signal to noise propertiesof the resulting images in these volumes.

[0122] Moreover, the γ-factor of carbon is about ¼ of the γ-factor forhydrogen resulting in a Larmor frequency of about 10 MHz at 1 T. Therf-absorption in a patient is consequently and advantageously less thanin ¹H imaging. A further advantage of MR imaging agents containingpolarised ¹³C nuclei is the ability to utilise large changes in chemicalshift in response to physiological changes, e.g. pH or temperature.

[0123] In one preferred embodiment, a “native image” of the sample (e.g.body) may be generated to provide structural (e.g. anatomical)information upon which the image obtained in the method according to theinvention may be superimposed. Such a native image is generally notavailable where the imaging nucleus is ¹³C due to the low naturalabundance of ¹³C in the body. Thus the native image may be convenientlyobtained as a proton MR image in an additional step to the method of theinvention.

[0124] The MR imaging agent may be conveniently formulated withconventional pharmaceutical or veterinary carriers or excipients. MRimaging agent formulations manufactured or used according to thisinvention may contain, besides the MR imaging agent, formulation aidssuch as are conventional for therapeutic and diagnostic compositions inhuman or veterinary medicine but will be clean, sterile and free ofparamagnetic, superparamagnetic, ferromagnetic or ferrimagneticcontaminants. Thus the formulation may for example include stabilizers,antioxidants, osmolality adjusting agents, solubilizing agents,emulsifiers, viscosity enhancers, buffers, etc. Preferably none of suchformulation aids will be paramagnetic, superparamagnetic, ferromagneticor ferrimagnetic. The formulation may be in forms suitable forparenteral (e.g. intravenous or intraarterial) or enteral (e.g. oral orrectal) application, for example for application directly into bodycavities having external voidance ducts (such as the lungs, thegastrointestinal tract, the bladder and the uterus), or for injection orinfusion into the cardiovascular system. However solutions, suspensionsand dispersions in physiological tolerable carriers (e.g. water) willgenerally be preferred.

[0125] For use in in vivo imaging, the formulation, which preferablywill be substantially isotonic, may conveniently be administered at aconcentration sufficient to yield a 1 micromolar to 1M concentration ofthe MR imaging agent in the imaging zone; however the preciseconcentration and dosage will of course depend upon a range of factorssuch as toxicity, the organ targeting ability of the MR imaging agent,and the administration route. The optimum concentration for the MRimaging agent represents a balance between various factors. In general,optimum concentrations would in most cases lie in the range 0.1 mM to10M, especially 0.2 mM to 1M, more especially 0.5 to 500 mM.Formulations for intravenous or intraarterial administration wouldpreferably contain the MR imaging agent in concentrations of 10 mM to1M, especially 50 mM to 500 mM. For bolus injection the concentrationmay conveniently be 0.1 mM to 10M, preferably 0.2 mM to 10M, morepreferably 0.5 mM to 1M, still more preferably 1.0 mM to 500 mM, yetstill more preferably 10 mM to 300 mM.

[0126] Parenterally administrable forms should of course be sterile andfree from physiologically unacceptable agents and from paramagnetic,superparamagnetic, ferromagnetic or ferrimagnetic contaminants, andshould have low osmolality to minimize irritation or other adverseeffects upon administration and thus the formulation should preferablybe isotonic or slightly hypertonic. Suitable vehicles include aqueousvehicles customarily used for administering parenteral solutions such asSodium Chloride solution, Ringer's solution, Dextrose solution, Dextroseand Sodium Chloride solution, Lactated Ringer's solution and othersolutions such as are described in Remington's Pharmaceutical Sciences,15th ed., Easton: Mack Publishing Co., pp. 1405-1412 and 1461-1487(1975) and The National Formulary XIV, 14th ed. Washington: AmericanPharmaceutical Association (1975). The compositions can containpreservatives, antimicrobial agents, buffers and antioxidantsconventionally used for parenteral solutions, excipients and otheradditives which are compatible with the MR imaging agents and which willnot interfere with the manufacture, storage or use of the products.

[0127] Where the MR imaging agent is to be injected, it may beconvenient to inject simultaneously at a series of administration sitessuch that a greater proportion of the vascular tree may be visualizedbefore the polarisation is lost through relaxation. Intra-arterialinjection is useful for preparing angiograms and intravenous injectionfor imaging larger arteries and the vascular tree.

[0128] The dosages of the MR imaging agent used according to the methodof the present invention will vary according to the precise nature ofthe MR imaging agents used, of the tissue or organ of interest and ofthe measuring apparatus. Preferably the dosage should be kept as low aspossible whilst still achieving a detectable contrast effect. Typicallythe dosage will be approximately 10% of LD₅₀, eg in the range 1 to 1000mg/kg, preferably 2 to 500 mg/kg, especially 3 to 300 mg/kg.

[0129] Once the MR imaging agent has been administered to the subject,the chosen procedures for detecting MR signals are that which is wellknown from conventional MR scanning. It is advantageous to use fastsingle shot imaging sequences e.g. EPI, RARE or FSE.

[0130] In conventional ¹H-nmr imaging, the polarization which isresponsible for the MR signal derives from the equilibrium polarizationat the magnetic field of the primary magnet of the MR imaging apparatus.After an imaging sequence, this polarization (the magnetization in the zdirection) is recovered by T₁ relaxation. By contrast where the MRsignal derives from hyperpolarization of the reporter nuclei (e.g. ¹³C,³He, ¹²⁹Xe, ¹⁵N, ²⁹Si, etc), the hyperpolarization cannot be recoveredand the MR signal following a 90° RF pulse must be recovered by a trainof 180° RF pulses.

[0131] Where however the hyperpolarization results from hydrogenationwith parahydrogen the magnetization in the z direction is split into twopopulations with opposite signs (polarities) of magnetization, +M_(o)and −M_(o). In a preferred imaging sequence, after an 90° RF pulse, the180° RF refocussing pulses should be timed such that the two componentsare parallel (in phase) at the echo time. This can be achieved by aninitial delay of Δτ+τ between the 90° RF pulse and the first 180° RFrefocussing pulse with the subsequent 180° RF pulses occurring at a timeseparation TE=2τ. Δτ here has the value 1/(2J) where J is the couplingconstant for the reporter nucleus. A total of N 180° RF pulses will berequired where N is the image matrix size in the phase-encodingdirection. Signal detection occurs between the 180° RF pulses. Due tothe coupling constant J, there are limitations on the length of thesampling time—if unwanted modulations, and hence ghosting in the phasedirection of the image are to be avoided, the sampling time should notexceed 1/(4J). Typically the sampling time will be 1 to 8 ms. Theinter-echo time should exceed the sampling time as little as technicallypossible to ensure maximum signal to noise. A schematic illustration ofthis imaging sequence is shown in FIG. 9.

[0132] In a standard CPMG-sequence, the 180° RF pulses are phase shiftedΠ/2 relative to the 90° RF pulse, e.g. 90°×−180° y−180° y . . . ; thisarrangement is preferred for the imaging sequence described above.

[0133] Thus using an initial focussing delay makes it feasible to imagea contrast agent with two anti parallel resonance lines as would beachieved by hydrogenation with para-hydrogen.

[0134] In an alternative approach, the problem can be addressed byapplying 180° RF pulses (180° RF_(H)) at the proton frequencysimultaneously with the 180° RF pulses (180° RF_(x)) at the reporternucleus frequency. The effect of the 180° RF_(H) pulse is to change thesign of the J-coupling so that this is not refocussed by the 180° RF_(x)pulse. The echo signals from the two magnetization components willprogressively begin to reinforce rather than cancel each other out andafter sufficient such 180° RF_(H) and 180° RF_(x) pulses, the twomagnetization components will be parallel (in phase). Thereafter nofurther 180° RF_(H) pulses are required. The two components +M_(o) and−M_(o) will be in phase after time T=1/(2J). If the spacing between the180° RF_(H) pulses is 2τ then the nubmer of 180° RF_(H) pulses requiredis n where 2Πτ=1/(2J), ie. n=¼Jτ). τ can be selected such that n is anintegral number. Alternatively put, TE is set to 1/(2nJ).

[0135] In a standard CPMG-sequence, the 180° RF pulses are phase shiftedΠ/2 relative to the 90° RF pulse. In the imaging sequence discussedabove, which is a derivative of a RARE sequence, the 180° RF_(x) pulsesare of the same phase as the 90° RF_(x) pulse.

[0136] Using this sequence, illustrated schematically in FIG. 17, thelongitudinal magnetization is turned to the xy plane by a single 90°RF_(x) pulse at the beginning of the sequence. Thus the fullmagnetization of the hyperpolarized reporter nuclei is available forgenerating an image. Compared to sequences using a train of low flipangle RF pulses the gain in signal to noise is approximately an order ofmagnitude. Moreover a signal may be obtained from a system with twoantiparallel resonance lines, without need for asymmetric echoes (ie.where spin echoes and gradient echoes are not aligned). This isadvantageous since the use of asymmetric echoes makes the imagingsequence sensitive to magnetic field inhomogeneities and results inimage artefacts.

[0137] Where a sequence based on a gradient echo pulse sequence andultra low flip angles for RF pulses, the most commonly used sequence forhyperpolarized noble gases, is used for para-hydrogen hyperpolarizedreporter nuclei, an echo time TE of 1/(2J) is required, resulting in atotal acquisition time of N/2J where N is the image matrix sign. Where n180° RF_(H) pulses are used to change the size of the J coupling andprevent refocussing of the J-coupling, the echo time is 1/(2nJ) and theimage acquisition time N/(2nJ), ie. a reduction by a factor of n. Thisreduction in scan time is beneficial as it reduces the signal loss dueto T₂ relaxation. By way of example if the matrix size is 256 and theJ-coupling is 25 Hz then the scan time for a single slice is more than 5s if a gradient echo sequence is used. Where the imaging sequence ofFIG. 9 is used, the total acquisition time is N (2τ), which, dependingon the imager, can be reduced to for example 0.5 to 2.5 seconds. Byusing the RARE-derivative sequence of FIG. 17 discussed above, the scantime can be reduced to 2.5 s (n=2) or 1.7 s (n=3), etc. (RARE sequencesand sequences used in imaging hyperpolarized gases are described byHennig et al. in Magn. Reson. Med 3: 823-833 (1986) and Zhao et al. inNucl. Instrum. and Meth. in Phys. Res. A402: 454-460 (1998)).

[0138] The method of the invention may also be used for ¹H magneticresonance imaging using the hydrogen hyperpolarisation introduced bypara-hydrogen hydrogenation of an unsaturated bond. Here, imagingsequences which bring into phase the +Mo and −Mo magnetisationcomponents are desirably used and the unsaturated bond is desirablybetween atoms which remain bonded together in the resulting MR imagingagent.

[0139] The contents of all publications referred to herein are herebyincorporated by reference.

[0140] Embodiments of the invention are described further with referenceto the following non-limiting Examples and the accompanying drawings, inwhich:

[0141]FIG. 1 is a plot of polarization enhancement of reporter nuclei(in an AA′x spin system with J₁₂=10.65 Hz, J₁₃=0.3 Hz and J₂₃=15.5 Hz)against the magnetic field strength at which hydrogenation withpara-hydrogen enriched hydrogen occurs;

[0142]FIGS. 2 and 3 are reaction schemes for catalysed hydrogenation ofprecursor compounds;

[0143]FIG. 4 is a diagram of a phantom;

[0144] FIGS. 5 to 7 are simulated MR images of the phantom of FIG. 4;

[0145]FIG. 8 is a schematic representation of apparatus according to theinvention for hydrogenation in a magnetically shielded reaction zone;

[0146]FIG. 9 is a schematic representation of a RARE derivative imagingsequence;

[0147]FIGS. 10 and 11 are ¹³C MR images of the rat stomach;

[0148]FIG. 12 is a ¹H-MR image of the rat stomach;

[0149]FIG. 13 shows a superposition of a ¹³C-MR image of the rat stomachon a ¹H MR image of the same;

[0150]FIG. 14 is a ¹³C-MR image obtained using a standard RARE-sequenceof a phantom containing a constrast agent containing ¹³C at naturalabundance and hydrogenated by para-hydrogen at low (microtesla) field;

[0151]FIG. 15 is an image corresponding to that of FIG. 12 but wherehydrogenation was effected at earth field;

[0152]FIG. 16 is an image corresponding to that of FIG. 12 but wherehydrogenation was effected at earth field and where the imaging sequenceused is a modified RARE-derivative as discussed above; and

[0153]FIG. 17 is a schematic illustration of a modified RARE imagingsequence.

[0154] Referring to FIG. 8, the hydrogenation apparatus 1 comprises agenerally cylindrical glass reaction chamber 2, e.g. of 5 to 50 mminternal diameter, containing a bed 3 of glass beads defining a reactionzone and surrounded by thermostatted water jacket 4 having inlet 4 a andoutlet 4 b and four-layer magnetic shield 5. The reaction chamber isclosed at the top by a rubber septum 7 and is provided with an outletvalve 8 at its base. A para-hydrogen source (not shown) is attached to agas conduit 9 which can lead into the reaction chamber above or belowbed 3 depending on the position of valve 10. During hydrogenation,para-hydrogen may be vented from the reaction chamber through valve 11and outlet 12. Precursor compound and catalyst may be introduced intothe reaction chamber using a syringe 13 with a needle capable ofpiercing the septum.

[0155] Before use, water at 42° C. is circulated through the waterjacket for at least 10 minutes. Valve 11 is opened, and valve 10 is putin position to allow para-hydrogen flow into the reaction chamberthrough the lower (14) of inlets 14 and 15. Valve 8 is closed. Flow ofpara-hydrogen is commenced. A flow of 130 mL/min is suitable where thereaction chamber internal diameter is 15 mm and the beads are 3 mmdiameter. After 30 seconds, a solution of precursor and catalyst may beinjected through septum 7. After the hydrogenation reaction iscompleted, e.g. after 40 seconds, valve 10 is moved to directpara-hydrogen flow into the reaction chamber through upper inlet 15,valve 8 is opened and valve 11 is closed. The solution passing outthrough valve 8 is collected.

EXAMPLE 1

[0156] An experiment was carried out to compare the expected SNR in (1)He-images generated using helium at 1 atm in lung tissue, (2) ¹³C-imagesgenerated using hyperpolarised H₂ and (3) “standard” contrast enhancedproton images. All calculations were performed using MRI-simulationsoftware developed at Nycomed Innovation in Malmö Sweden. Thecalculation procedure is based on the k-space formalism (Petersson etal., 1993, Mag. Res. Imaging, 11: 557-568) and the multi dimensionaldescription (Petersson et al., 1997, Mag. Res. Imagin, 15: 451-467) ofthe image formation in MRI.

[0157] A mathematically defined phantom according to FIG. 4 was used toinput all calculations. The ¹³C was assumed to be in a bolus and themagnitude of the magnetization was raised to five times the magnitudeused for hydrogen. 50% polarisation was assumed and the concentrationwas 45.0 mM. The relaxation times for ¹³C were T₁=100 s and T₂=2 s. Theproton relaxation times are those found at 1.5 T. The blood containingcontrast agent uses the relaxation times found when the bolus trackingtechnique is utilized. Hyperpolarised helium was assumed to be in formof a gas at 1 atm and the relaxation times were chosen in accordancewith Bachert et al. Magn. Res. in Medicine, 36: 192-196 (1996) when thegas is present inside the lungs.

[0158] The short T₂ (T₂*) is due to the high diffusion coefficient (D=2cm² s⁻¹). The magnitude of the helium magnetization was raised to 16times that used for hydrogen. 50% polarisation was assumed and theconcentration was 45.0 mM.

[0159] Two different pulse sequences were used. A fast gradient echosequence, FLASH, was used to generate the hydrogen image and the heliumimage. The hydrogen pulse sequence parameters were TR/TE/α=8 ms/2 ms/30°and the helium pulse sequence was 8 ms/2 ms/3°. The enhancement gain ofthe He-magnetization is in this way divided during the imaging process.

[0160] A RARE (Fast Spin Echo) sequence was used to generate the ¹³Cimage. Eight interleaves were used in order to simulate the situationfound when imaging the heart using gating. The ¹³C magnetization behavedthe same way as the He magnetization i.e. no new magnetization wasgenerated due to T₁-relaxation during the imaging process. During thecalculation the ¹³C were modelled in the form of a bolus and between theinterleave in the pulse sequence the excited magnetization was replacedwith fresh magnetization. If a static object was imaged the sequencecould have been performed as a single shot sequence without (due to thelong T₂ value) any loss in signal amplitude.

[0161] Results

[0162] Hydrogen

[0163] In the proton image (FIG. 5), the helium and the ¹³C do not showup. The signal from the blood and contrast agent appears bright. Theshort TR and the relatively high flip angle makes the image stronglyT₁-weighted. The muscle and the blood without contrast agent appearsdark. The signal amplitude in the ROI was 129 and SNR=107.

[0164] Helium

[0165] In the He-image (FIG. 6) the proton and ¹³C do not show up. Thesignal from the helium appears bright and no background from othertissues are present. The short TR and the relatively low flip anglegenerated an image which in normal proton imaging would be considered asa spin density image. The signal amplitude in the ROI was 347 andSNR=289.

[0166] Carbon-13

[0167] In the ¹³C-image (FIG. 7) the protons and the helium do not showup. The signal from ¹³C appears bright and no background from othertissues is present. The selected RARE sequence may be consideredT₂-weighted. The image was generated using a multi shot technique but asingle shot version would (due to the long T₂-value) result in the samesignal amplitude. The signal amplitude in the ROI was 2605 and SNR=1737

[0168] Conclusion

[0169] The generated signal amplitude and SNR values indicate thealready recognised utility of helium as a contrast agent in lungimaging. If the gas was dissolved in blood the signal amplitude woulddrop considerably (Martin et al., J. Mag. Res. Imaging, 1997, 7,848-851). The ¹³C image indicated that when the polarisation of enrichedhydrogen is transferred to a ¹³C-atom in a suitable organic moleculeimages with high SNR may be generated. Due to the long T₁ and T₂, modernfast single shot sequences may be used. Whilst the ¹³C-fluid behaves asa bolus the long Tl will make it possible to reach the heart with only amoderate loss in signal amplitude even if it is administered by i.v.injection.

EXAMPLE 2

[0170] The following reactions are performed and produce the enhancementeffects mentioned.

[0171] (A) Ph—C≡CH+para-hydrogen and homogeneous rhodium catalyst(giving ¹H enhancement of about 200 and 20% conversion in about 20seconds).

[0172] (B) EtOOC—C≡C—COOEt+para-hydrogen and homogeneous rhodiumcatalyst, converting about 100% in about 20 seconds to the cis C═Cproduct and giving ¹³C enhancement of about ×500.

[0173] (C)R—CH═CH—COOH+para-hydrogen and a resin bound rhodium catalystin water, converting about 75% in 8 minutes to RCH₂CHCOOH (where R is Hor COOH).

EXAMPLE 3

[0174] Low-Field Enhancement of the Para-Hydrogen Signal

[0175] Acetylene dicarboxylic acid dimethyl ester (0.5 g) with a naturalabundance of ¹³C, and (bicyclo[2.2.1]hepta-2,5-diene)[1,4-bis(diphenylphosphino)butane] rhodium(I) tetrafluoroborate (0.12mmol) in a solution of deuteroacetone (5 ml) was hydrogenated withhydrogen gas enriched in para-hydrogen (50%) for 40 seconds with ajacket temperature of 42° C. in the hydrogenation reactor describedabove in connection with FIG. 8 with the magnetic screen in place.

[0176] The solution was transferred to an nmr-tube and, following a 90°pulse, a spectrum was recorded at the ¹³C frequency in a 7TNMR-spectrometer within 20 seconds after the reaction was finished. Theintensity of the signal was compared to a standard sample and was foundto be 1500 times the thermodynamic signal at 250 and 7T. It wasnecessary to detune the NMR-probe significantly to be able to performproper excitations on such a highly polarized sample.

[0177] In another experiment the sample solution was transferred to aglass vial and imaged using a standard RARE-sequence. The result isshown in FIG. 14. As a comparison a new sample was hydrogenated inambient field (80 micro-Tesla) and subjected to the same imaging scheme.No signal could be detected. The result is shown in FIG. 15.

EXAMPLE 4

[0178] Imaging of the Para-Hydrogen Enhanced Signal in Phantoms

[0179] Acetylene dicarboxylic acid dimethyl ester (6 mmol) with anatural abundance of ¹³C, and(bicyclo[2.2.1]hepta-2,5-diene)[1,4-bis(diphenylphosphino)butane]rhodium(I) tetrafluoroborate (0.23 mmol) in a solution of deuteroacetone(10 ml) was hydrogenated with hydrogen gas enriched in para-hydrogen(50%) for 40 seconds with a jacket temperature of 42° C. in thehydrogenation reactor described above in connection with FIG. 8 with themagnetic screen removed.

[0180] The sample was transferred to a vial and placed in the magnet ofan imaging magnet and a picture was recorded within 30 seconds after thereaction was finished. After Fourier transform, the image shown in FIG.16 was obtained and after calibration with a standard sample the signalenhancement was calculated to be 225 times the polarization obtained atequilibrium at 2.4 T and 20° C. The special pulse sequence describedabove and shown schematically in FIG. 9 was used (90×19.2 ms, 5 ms-(180y-10 ms)×64). The focusing delay was set to 19.2 ms and the inter-echodelay was set to 10 ms.

EXAMPLE 5

[0181] Imaging of the Para-Hydrogen Enhanced Signal in Rat

[0182] Acetylenene dicarboxylic acid dimethyl ester-1-¹³C (99%) (6mmol), and(bicyclo[2.2.1]hepta-2,5-diene)[1,4-bis(diphenylphosphino)butane]rhodium(I)tetrafluoroborate (0.23 mmol) in a solution of deuteroacetone (10 ml)was hydrogenated with hydrogen gas enriched in para-hydrogen (50%) for40 seconds with a jacket temperature of 42° C. in the hydrogenationreactor described above in connection with FIG. 8 with the magneticscreen removed.

[0183] The hydrogenated sample was transferred to a syringe and injectedinto the stomach of a rat. The rat was then placed in the imaging magnetand a picture was recorded using the same pulse sequence as above. As areference, the proton image of the rat in the same position was alsoobtained. A control experiment where the pulse sequence was repeatedafter relaxation of the contrast agent was also performed. No imagecould be detected in this case. The results are shown in Figures 10 to13.

1. A method of magnetic resonance investigation of a sample, said methodcomprising: (i) reacting para-hydrogen enriched hydrogen with ahydrogenatable MR imaging agent precursor containing a non-hydrogen nonzero nuclear spin nucleus to produce a hydrogenated MR imaging agent;(ii) administering said hydrogenated MR imaging agent to said sample;(iii) exposing said sample to radiation of a frequency selected toexcite nuclear spin transitions of said non-zero nuclear spin nucleus insaid hydrogenated MR imaging agent; (v) detecting magnetic resonancesignals of said non-zero nuclear spin nucleus from said sample; and (vi)optionally, generating an image or biological functional data or dynamicflow data from said detected signals.
 2. A method as claimed in claim 1wherein said enriched hydrogen has a more than 45% proportion ofpara-hydrogen.
 3. A method as claimed in claim 1 wherein said enrichedhydrogen has a more than 90° proprtion of para-hydrogen.
 4. A method asclaimed in any of the preceding claims wherein said MR imaging agentprecursor contains nuclei selected from F, Li, C, N, Si and P nuclei. 5.A method as claimed in claim 4 wherein said non-zero nuclear spinnucleus is selected from ¹³C, ¹⁵N and ²⁹Si.
 6. A method as claimed inclaim 5 wherein said non-zero nuclear spin nucleus is ¹³C.
 7. A methodas claimed in any one of claims 1 to 6 wherein said non-zero nuclearspin nucleus is present at a level greater than its natural isotopicabundance.
 8. A method as claimed in any one of claims 1 to 7 whereinsaid precursor contains a hydrogenatable unsaturated carbon-carbon bond.9. A method as claimed in claim 8 wherein said non-zero nuclear spinnucleus is present in said precursor one or two bonds distant from saidunsaturated bond.
 10. A method as claimed in claim 9 wherein saidnucleus one or two bonds distant from said unsaturated bond is onlydirectly bonded to atoms which in their predominant isotopic state havezero nuclear spin.
 11. A method as claimed in any one of claims 1 to 10wherein in said MR imaging agent the coupling constant (J) between saidnon-zero spin nucleus and a proton deriving from para-hydrogen isbetween 10 and 100 Hz.
 12. A method as claimed in claim 11 wherein thenmr signal from said non-zero nuclear spin nucleus in said MR imagingagent has a line width of less than 10 Hz.
 13. A method as claimed inclaim 12 wherein said MR imaging agent has a molecular weight of lessthan 500D.
 14. A method as claimed in any one of claims 1 to 13 whereinsaid MR imaging agent is water-soluble.
 15. A method as claimed in anyone of claims 1 to 14 wherein the chemical shift and/or the couplingconstant of said non-zero nuclear spin nucleus in said MR imaging agentis sensitive to the physicochemical environment of said agent.
 16. Amethod as claimed in claim 15 wherein said non-zero nuclear spin nucleusin said MR imaging agent is sensitive to pH and wherein said signals aremanipulated to produce an image or data indicative of pH.
 17. A methodas claimed in any one of claims 1 to 16 wherein step (i) is effected ina magnetic field smaller than the earth's ambient field.
 18. A method asclaimed in claim 17 wherein step (i) is effected in a magnetic field ofless than 10 μT.
 19. A method as claimed in any one of claims 1 to 18wherein in steps (iii) and (iv) said sample is exposed to a 90° pulse ofradiation of a frequency selected to excite nuclear spin transitions ofsaid non-zero nuclear spin nucleus followed by 180° pulses of saidradiation, where the time interval between said 180° pulses is 2τ andthe time interval between said 90° pulse and the subsequent 180° C.pulse is τ plus Δτ where Δτ is 1/(2J) where J is the coupling constantof said non-zero nuclear spin nucleus in said MR imaging agent.
 20. Amethod as claimed in any one of claims 1 to 18 wherein in steps (iii)and (iv) said sample is exposed to a 90° pulse of radiation of afrequency selected to excite nuclear spin transitions of said non-zeronuclear spin nucleus followed at time intervals of 21 by 180° pulses ofsaid radiation of the same phase and where for the first n said 180°pulses said sample is simultaneously exposed to 180° pulses of radiationof a frequency selected to excite proton nuclear spin transitions, therelation between n and τ being τ=1/(4nJ) where J is the couplingconstant of said non-zero nuclear spin nucleus in said MR imaging agent.21. A method as claimed in any one of claims 1 to 19 wherein step (i) iseffected using a rhodium-based hydrogenation catalyst.
 22. Use ofpara-hydrogen enriched hydrogen in the manufacture of an MR imagingagent for non-proton MR imaging of a sample.
 23. Use of para-hydrogenenriched hydrogen in MR imaging of a sample.
 24. Use as claimed ineither one of claims 22 and 23 wherein said imaging is ¹³C NMR imagingof a sample.
 25. A precursor compound: (i) containing a hydrogenatableunsaturated bond; (ii) containing a non-hydrogen non zero nuclear spinnucleus at greater than natural isotopic abundance; (iii) having amolecular weight below 1000OD; and (iv) which following hydrogenationhas an nmr spectrum for said non-hydrogen non zero nuclear spin nucleuswhich is a multiplet having a coupling constant relative to one of thehydrogens introduced by hydrogenation of 1 to 300 Hz and having alinewidth of less than 100 Hz, and wherein when said precursor compoundis a ¹³C enriched compound then said nucleus is a quaternary carbonnucleus.
 26. A compound as claimed in claim 25 containing the followingmolecular sub-units: (i) at least one C═C or C≡C bonds; (ii) a C, N orSi atom separated by one or two bonds from a C═C or C≡C bond, bound onlyto atoms the naturally most abundant isotope form of which has a nuclearspin I=0, and not coupled by a series of covalent bonds to any atoms thenaturally most abundant isotopic form of which has I>½; and (iii) atleast one water-solubilizing moiety, ie. a functional group whichimparts water solubility to the molecule.
 27. A reporter compound: (i)containing at least two protons; (ii) containing a non-hydrogen non zeronuclear spin nucleus at greater than natural isotopic abundance; (iii)having a molecular weight below 1000D; and (iv) having an nmr spectrumfor said non-hydrogen non zero nuclear spin nucleus which is a multiplethaving a coupling constant relative to one of said at least two protons1 to 300 Hz and having a linewidth of less than 100 Hz, and wherein whensaid reporter compound is a ¹³C enriched compound then said nucleus is aquaternary carbon nucleus.
 28. A compound as claimed in claim 27containing the following molecular sub-units: (i) at least one CH—CH orCH═CH moiety; (ii) a C, N or Si atom separated by one or two bonds froma CH—CH or CH═CH moiety, bound only to atoms the naturally most abundantisotopic form of which has I=0, and not coupled by a series of covalentbounds to any atoms the naturally most abundant isotopic form of whichhas I>½; and (iii) at least one water-solubilizing moiety, ie. afunctional group which imparts water solubility to the molecule.
 29. Aphysiologically tolerable MR imaging agent composition comprising an MRimaging agent together with one or more physiologically tolerablecarriers or excipients, said imaging agent containing non-hydrogennuclei having a nuclear spin of ½, characterised in that said nuclei arepolarized such that their nmr signal intensity is equivalent to a signalintensity achievable in a magnetic field of at least 0.1T.
 30. Acomposition as claimed in claim 29 wherein said nuclei are polarizedsuch that their nmr signal intensity is equivalent to a signal intensityachievable in a magnetic field of at least 100T.
 31. A composition asclaimed in either one of claims 29 and 30 wherein said nucleus ispresent in an amount in excess of its natural isotopic abundance.
 32. Acomposition as claimed in claim 31 wherein said nucleus is present in aselected position in the molecular structure of said agent at anabundance of at least 50%.
 33. A composition as claimed in any one ofclaims 29 to 32 wherein T₁ for said nuclei at earth's magnetic field andat ambient temperature is at least 1 second.
 34. An apparatus forhydrogenation comprising: a reaction chamber having therein a reactionzone, said reaction chamber having a gas inlet and a gas outlet; atemperature controller arranged to control the temperature in saidreaction zone; and magnetic shielding arranged about said reaction zoneand sufficient to cause the magnetic field in said reaction zone to beless than 10 μT.
 35. Apparatus as claimed in claim 34 wherein saidreaction zone contains a particulate bed and said reaction chamber isprovided with a liquid outlet below said bed and a liquid inlet abovesaid bed.
 36. Apparatus as claimed in claim 35 comprising: (i) areservoir of para-hydrogen enriched hydrogen; (ii) a reaction chamberhaving a reaction zone containing a particulate bed and having a firstgas inlet below said bed, a first gas outlet above said bed, a solutioninlet above said bed and a solution outlet below said bed; (iii) a gasconduit from said reservoir to said first gas inlet in the reactionchamber; (iv) a temperature controller disposed around said reactionchamber at at least said reaction zone; and (v) a magnetic shielddisposed around said reaction chamber at at least said reaction zone.37. The use of para-hydrogen enriched hydrogen in the manufacture of anMR imaging agent for use in a method of diagnosis involving generationof an MR image by non-proton MR imaging.
 38. The use of a hydrogenatablecompound containing a non hydrogen non zero nuclear spin nucleus in themanufacture of an MR imaging agent for use in a method of diagnosisinvolving generation of an MR image by non-proton MR imaging, saidmanufacture involving hydrogenation of said compound with para-hydrogenenriched hydrogen.